Cloning and expression analysis of BAG-1 gene in Epinephelu coioides

【Objective】To investigate the structural characteristics，tissue expression distribution of BAG-1 gene （EcBAG-1）in Epinephelu coioides and its expression changes of it after stimulation of lipopolysaccharide（LPS）and polyinosinic acid（PolyI∶C），with an aim to further reveal the mechanism of fish BAG-1 in pathogen infection，so as to provide a reference for understanding the immunity of fish against disease.【Method】The EcBAG-1 gene was cloned by designing primers based on its EST sequence，and bioinformatics analysis was performed by using online software such as SMART，TMHMM Server v.2.0，SOPMA，SWISS-MODEL，ExPASy，ClustalX and MEGA 5.0. The tissue expression characteristics of EcBAG-1 gene and its expression pattern after LPS and Poly I:C stimulation were detected by using realtime fluorescence quantitative PCR.【Result】The open reading frame（ORF）of the cloned EcBAG-1 gene was 624 bp in length，encoding 207 amino acids，and EcBAG-1 protein molecular weight was 49.84 kD，theoretical isoelectric point （pI）was 5.18 and it was a hydrophobic protein. Ec-BAG-1 protein had a conserved BAG domain and an UBQ ubiquitinrelated domain of the BAG-1 family，and no transmembrane domain，it mainly existed in the cytoplasm and nucleus. In the secondary structure，α - helix accounted for 65.22%，extended chain 10.63%，irregular coiled coil 20.29% and β - turned angle only 3.86%. EcBAG-1 amino acid sequence of E. coioides had the highest similarity（96.62%）with Larimichthys crocea，and the two species was the most closely related. E. coioides was relatively distantly related to Gallus gallus and Xenopus tropicalis，with corresponding BAG-1 amino acid sequence similarities of 58.45% and 57.50%，respectively. EcBAG-1 gene was expressed in liver，thymus，spleen，head kidney，gill，skin，muscle，brain，peripheral blood lymphocytes and heart of E. coioides，and the relative expression was highest in brain，which was significantly higher than that in other tissues（P>0.05，the same below）. After 24 h of LPS and PolyI:C stimulation，relative expression of EcBAG-1 gene in the spleen of E. coioides.【Conclusion】EcBAG-1 protein contains the typical BAG structural domain and UBQ structural domain of the BAG-1 protein family，and it has biological function similar to mammalian BAG-1 of mammals，which means EcBAG-1 gene plays an important role in the resistance to bacterial/viral infection in E. coioides， broadening the idea of understanding the anti-infection mechanism in fish.